The main goal of this application is to characterize the process involved in agonist-stimulated synthesis of anandamide (arachidonylethanolamine) in intact cells. A preliminary report (8) from this laboratory showed that agents capable of activating phospholipases to release free arachidonic acid appear to also promote anandamide synthesis. This observation has provided the basis for the hypothesis that we wish to test in this proposal, namely, that physiological control of anandamide synthesis resides in the regulation of cellular phospholipase activity. This is the situation for the other members of the eicosanoid family of which anandamide is a novel member. Anandamide has been suggested to be a putative endogenous cannabimimetic substance, however, its significance will probably extend beyond the area of drug abuse. Indeed, it has been speculated that it may represent a class of novel neuroregulatory agents because of its distribution and biological effects. Receptors that bind anandamide have also been observed in peripheral cells associated with the immune system suggesting that it may have functions outside of the central nervous system as well. Thus, an understanding of how its tissue levels are controlled becomes a matter of some importance. Specific questions to be addressed in this proposal are; (a) What are the agonists that are able to stimulate anandamide synthesis? (b) Are cells in culture suitable experimental models for such studies; (c) Are receptors required for synthesis in intact cells? (d) Which enzymic mediators are responsible for anandamide synthesis? (e) What are the important precursors for cellular anandamide synthesis? Based on what is known about anandamide's properties, its significance may be that it is involved in novel neuroregulatory processes that control CNS functions such as mood, motor coordination, cognitive ability, time-sense and self-perception. In addition, peripheral effects on the cardiovascular, immune, endocrine and respiratory systems may also be part of anandamides' spectrum of activities. The proposed studies may provide useful models for the development of therapeutic approaches to diseases such as multiple sclerosis, Huntington's, etc. where the use of cannabinoids has been explored. Methods for controlling endogenous anandamide synthesis in vivo could prove to be beneficial in relieving the symptoms of these types of ailments. Finally, it is possible that anandamide is only one member of a family of regulatory agents and that data resulting from this project will provide evidence for the existence of other more potent or specific "anandamides".